Branching morphogenesis of the embryonic kidney occurs by a tightly regulated interaction between the ureteric bud epithelium and metanephric mesenchyme. Disruption of mechanisms regulating this process can result in defects ranging from complete agenesis to a variety of malformations compromising adult renal function. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid influencing cellular proliferation, apoptosis, migration, and differentiation. S1P regulatory enzymes and S1P receptors exhibit developmentally regulated expression and are critical to developmental processes in other systems. The innovative aspect of this proposal lies in the fact that S1P signaling is closely integrated with growth factors known to influence branching morphogenesis, yet the role of S1P in this process has not been addressed. Our laboratory has focused its research efforts on determining the influence of S1P signaling on kidney development. The results of this work have led us to an understanding that sphingosine kinase activity and S1P receptor signaling are essential for tubulogenesis and that S1P signaling provides a previously unrecognized mechanism contributing to the elaborate process of renal morphogenesis. The long- term goal of this research is to define mechanisms by which S1P signaling mediates kidney development. In this proposal, we will: 1) link activity of S1P regulatory enzymes and spatiotemporal expression of S1P receptors to early morphogenic events of kidney development using enzyme activity assays, real-time RT-PCR, and immunohistochemistry; and 2) demonstrate interaction with growth factors known to regulate branching of the ureteric bud using in vitro model systems including the metanephric kidney organ culture system and cell culture based systems of both ureteric bud epithelia and metanephric mesenchyme. Results from these studies will elucidate the basis of mechanisms that regulate S1P signaling in the developing kidney and contribute to the fundamental understanding of mechanisms regulating kidney development. Defective mechanisms regulating kidney morphogenesis can result in congenital abnormalities and nephron deficiency leading to hypertension and renal failure. Characterization of S1P signaling in the developing kidney will provide novel mechanistic insight to the process of branching morphogenesis, linking activities of previously identified factors with S1P into an integrated model that will advance our understanding of kidney development and elucidate potential sites of dysfunction responsible for developmental abnormalities. [unreadable] [unreadable] Significance: This is a highly significant project, as the sphingosine pathway is increasingly being acknowledged to be of great importance, yet has been minimally explored in organ development. [unreadable] [unreadable] Approach: Preliminary data include demonstrations that an inhibitor of sphingosine kinase (SK) inhibited branching in organ culture, addition of S1P could ameliorate the inhibition of SK effect on branching. These results provide sufficient justification for an R21 level grant aimed at further dissecting the role of sphingosine signaling in kidney development. [unreadable] [unreadable] Specific Aim 1 will further characterize the levels and spatial localization of SK and S1Prs in the developing kidney. These studies are appropriate. The only feasibility issue is whether appropriate antibodies are available, but if not, this can be done by in situ hybridization. For some of the genes in question, LacZ reporter knock-in mice are available as well. Additional studies will use selective receptor inhibitory agents to study the effect on branching. While pharmacological inhibitors are always problematic with regard to specificity, it is also true that the specificity of many of these agents has been dramatically improved in recent years, so that these sorts of studies can no longer be categorically dismissed. In this case they seem well justified. [unreadable] [unreadable] Specific Aim 2 will examine the possibility that known growth factor signaling pathways in the kidney, such as GDNF/c-Ret, are modified by Sphingosines. This is also a novel possibility that is worth examining. Both in vivo and in vitro studies are proposed, and seem feasible and appropriate. [unreadable] [unreadable] Investigators: Dr. Arend is a renal pathologist at the University of Cincinnati, which is a great center for developmental biology research. She has pioneered the study of sphingoisines in kidney development and is ideally suited to carry out the proposed research. [unreadable] [unreadable] Environment: The University of Cincinnati is an outstanding scientific environment, especially for developmental biology. [unreadable] [unreadable] Innovation: These studies are highly innovative. [unreadable] [unreadable] [unreadable]